Composition and Method for Treating Tinnitus

ABSTRACT

A method of treating tinnitus includes the step administering a composition to the mammal, wherein the composition consists essentially of a biologically effective amount of vitamin A, vitamin E, vitamin C, a vasodilator comprising magnesium, and, optionally, a withanolide, and/or resveratrol.

RELATED APPLICATIONS

This patent application is a continuation-in-part of U.S. patentapplication Ser. No. 13/839,760 filed on Mar. 15, 2013, which is acontinuation-in-part of U.S. patent application Ser. No. 13/091,931,filed Apr. 21, 2011, which is a continuation of U.S. application Ser.No. 12/761,121 filed on Apr. 15, 2010, which is a continuation of U.S.application Ser. No. 11/623,888, filed on Jan. 17, 2007. U.S.application Ser. No. 11/623,888 is now U.S. Pat. No. 7,951,845, and U.S.application Ser. No. 11/623,888 claims priority to and all advantages ofU.S. Provisional Patent App. Ser. No. 60/760,055, which was filed onJan. 19, 2006. U.S. patent application Ser. No. 13/839,760 is also acontinuation of U.S. application Ser. No. 13/091,931 filed on Apr. 21,2011, which is a continuation of U.S. application Ser. No. 12/761,121filed on Apr. 15, 2010, which is a continuation of U.S. application Ser.No. 11/623,888, filed on Jan. 17, 2007. U.S. application Ser. No.11/623,888 is now U.S. Pat. No. 7,951,845, and U.S. application Ser. No.11/623,888 claims priority to and all advantages of U.S. ProvisionalPatent App. Ser. No. 60/760,055, which was filed on Jan. 19, 2006.

GOVERNMENT LICENSE RIGHTS

This disclosure was made with government support under DC004058 awardedby the National Institutes of Health. The government has certain rightsin the disclosure.

BACKGROUND OF THE DISCLOSURE

1. Field of the Disclosure

The present disclosure generally relates to method for treatingtinnitus. More specifically, the method includes administering acomposition to the mammal, wherein the composition consists essentiallyof a biologically effective amount of vitamin A, vitamin E, vitamin C, avasodilator comprising magnesium, and, optionally, a withanolide, and/orresveratrol.

2. Description of the Related Art

Extensive studies have been performed on compositions for treatingvarious types of hearing loss/damage. Tinnitus, typically described asthe hearing of a ringing noise, can be perceived in one or both earsand/or in the head. In some cases, tinnitus may be described ashigh-pitched whining, electric buzzing, hissing, humming, or whistling.Tinnitus can alternatively be described as ticking, clicking, roaring,“crickets” or “tree frogs” or “cicadas.” In some cases, tinnitus may bedescribed as hearing tunes, songs, beeping, sizzling, sounds thatresemble human voices, or even as a steady tone that resembles the toneheard during a hearing test. Tinnitus has also been described as a“whooshing” sound because of acute muscle spasms, as of wind or waves.Tinnitus can be intermittent or it can be continuous. In still otherinstances, the sound may range from a quiet background noise to one thatcan be heard even over loud external sounds. A specific type oftinnitus, known as pulsatile tinnitus, includes hearing the sounds ofone's own pulse or muscle contractions. Some patients with tinnitus havesome degree of hearing loss and can be unable to clearly hear externalsounds that occur within the same range of frequencies as the tinnitussounds. This has led to the suggestion that one cause of tinnitus mightbe a homeostatic response of central dorsal cochlear nucleus auditoryneurons that makes them hyperactive in compensation to auditory inputloss.

Tinnitus can occur spontaneously and can be identified by defaultwithout a known cause. However, tinnitus has been most commonlyassociated with noise-induced hearing loss (NIHL). By some estimates,tinnitus results from NIHL about 80% of the time. High intensity noiseis an environmental stress factor for the ear that causes damage whichcan lead to cell death. High intensity noise can cause damage tomicromechanical properties of sensory transducers in the ear, can causechanges in blood flow in the ear, can cause modification inintracellular ion transport properties, can cause depletion of sensorycell transmitter substances (e.g. glutamate), can cause changes in postsynaptic membrane transmitter receptors (e.g. gluR) on afferent nervefibers, can cause modification of dispersion and uptake properties oftransmitters in extracellular, synaptic spaces, and/or can cause changesin postsynaptic membrane biophysical properties that may affect space-and time-constant properties modifying depolarization. In addition, highintensity noise can cause changes of an excitotoxic nature inpostsynaptic membranes causing destruction of afferent neural tissues.Any one or more of these changes can result in modification ofspontaneous activity in individual or small populations of afferentnerve fibers, which can result in the perception of tinnitus. While anumber of factors may influence these changes, intercellular redoxproperties of cells, blood flow to the inner ear, and calcium uptake bypostsynaptic afferent nerve fiber membranes may be of particularimportance in causing these changes.

Some agents have been tested for efficacy against NIHL and theirrelative efficacy has been found to differ from their relative efficacyfor drug-induced tinnitus. For example, allopurinol tends to beineffective in reducing gentamicin-induced ototoxicity, but may beeffective in reducing noise-induced tinnitus. Accordingly, there remainopportunities to develop effective methods of treating various types andcauses of tinnitus. There is also an opportunity to provide acomposition for treating tinnitus.

SUMMARY OF THE DISCLOSURE

The subject disclosure provides a method of treating tinnitus thatincludes the step administering a composition to the mammal, wherein thecomposition consists essentially of a biologically effective amount ofvitamin A, vitamin E, vitamin C, a vasodilator comprising magnesium,and, optionally, a withanolide, and/or resveratrol.

DETAILED DESCRIPTION

A composition for treating tinnitus includes components that mayfunction through different biological mechanisms to provide an additiveeffect that is equal to or greater than a sum of the effect of theindividual components. The composition is typically used for treatingtinnitus that may result from trauma to an inner ear of a mammal or mayhave no known cause. The trauma may be further defined asmechanically-induced metabolic trauma, mechanical/metabolic trauma,stress trauma, stress-induced damage, or environmental stress. However,it is also possible that the composition may also be used to treat orprevent other types of tinnitus, including, for example, age-relatedtinnitus, antibiotic-induced tinnitus, and chemotherapeutic-inducedtinnitus. The composition may further be used to prevent tinnitus duringrestoration surgery performed on the inner ear.

A method in accordance with the instant disclosure includes the step ofadministering a composition to a mammal that includes components thatfunction through different biological mechanisms. In the method, thecomposition is typically used for treating tinnitus resulting fromtrauma to the inner ear of a mammal.

In various embodiments, the trauma may be further defined as highintensity noise. High intensity noise can cause damage tomicromechanical properties of sensory transducers in the ear, can causechanges in blood flow in the ear, can cause modification inintracellular ion transport properties, can cause depletion of sensorycell transmitter substances (e.g. glutamate), can cause changes in postsynaptic membrane transmitter receptors (e.g. gluR) on afferent nervefibers, can cause modification of dispersion and uptake properties oftransmitters in extracellular, synaptic spaces, and/or can cause changesin postsynaptic membrane biophysical properties that may affect space-and time-constant properties modifying depolarization. In addition, highintensity noise can cause changes of an excitotoxic nature inpostsynaptic membranes causing destruction of afferent neural tissues.Any one of these changes can result in modification of spontaneousactivity in individual or small populations of afferent nerve fibers,which can result in the perception of tinnitus. While a number offactors may influence these changes, intercellular redox properties ofcells, blood flow to the inner ear and calcium uptake by postsynapticafferent nerve fiber membranes may be of particular importance incausing these changes.

It has been found that one result of noise trauma, or other stressorssuch as age and drugs, is that free radicals form in association withmetabolic trauma. The free radicals damage sensitive structures, such ashair cells, within the ear and can initiate processes that can lead totinnitus. Vasoconstriction also occurs as a result of the noise, whichleads to decreased blood flow to the inner ear and causes cell deaththat results in tinnitus. It has been found that the underlying cause ofvasoconstriction is noise-induced free radical formation. Specifically,one of the molecules formed in the inner ear as a result of the presenceof free radicals is 8-isoprostane-2F alpha, which is a bioactive agent.The bioactive agent induces a constriction of blood vessels in the innerear, which causes a reduction in blood flow. In order to counteract thefree radical formation and the vasoconstriction, the composition of thesubject disclosure typically includes at least one scavenger of singletoxygen, a donor antioxidant, a third antioxidant, and a vasodilator.Unexpectedly, it was found that the composition including the at leastone scavenger of singlet oxygen, the donor antioxidant, the thirdantioxidant, and the vasodilator produce an additive effect that is notonly greater than the effect of any one of those components alone, butat least equal to or greater than a sum of the effects of each of thecomponents.

Many genetic hearing disorders are dependent upon a defect in a singlegene leading to hearing impairment or deafness; many reflect defects inmore that one gene, and may be associated with other, non hearing,clinical disorder. Tinnitus may be associated with either class ofgenetic hearing disorders and thus associated with one (non-syndromic)mutation or with mutations in more than one gene (e.g. syndromic) andmay be associated with other inherited clinical disorders

Tinnitus may reflect a defect in a gene resulting in flawed gene copieswhich mis-instructs production of a protein important for homeostasisand transduction processes in the inner ear. This could lead todisruption of micromechanical properties of hair cells, depolarizationdefects, inadequate production and or assembly of transmittersubstances, and/or compromised synaptic activity. This may also resultin disruption of ion metabolism, e.g. potassium, their distribution andmovement in the inner ear and particularly in the lateral wall andsensory cells, and a disruption of cellular homeostasis in these cells,frequently leading to cell death.

Since potassium homeostasis in the inner ear is important for normalfunction of the sensory cells of the inner ear (hair cells), anydisruption tends to result in hearing impairment and may inducetinnitus. Knockout mouse models of genetic hearing impairment may showearly widespread degeneration of both inner and outer hair cells,presumably secondary to defects that upregulate apoptotic cell deathpathways. In humans, hearing impairment may begin sometime followingbirth and progress until profound deafness occurs. However, gene defectsmay be expressed as mild to moderate hearing impairment.

More specifically, one or more genetic defects may lead to disruption ofpotassium homeostasis in an inner ear of a mammal. Potassium homeostasismay regulate apoptosis such that stress-driven disruption of inner earpotassium homeostasis may lead to increase production of free radicalsby mitochondria directly leading to upregulation of apoptotic cell deathpathways as well as support direct potassium induced cytochrome crelease and apoptosis. Potassium channels Kv1.3, mitochondrial Ca²⁺regulated potassium channel, mitoBKCa, and mitochondrial ATP-regulatedpotassium channel—mitoKATP have been demonstrated in mitochondrialmembranes. Mitochondrial potassium channels effect energy production bythe mitochondrion. In addition, there may be a direct dependence of freeradical formation on potassium channel function during the respiratorychain in mitochondrial function. Moreover, increased mitochondrial K+influx may result in release of cytochrome c and caspase-3 followed byapoptosis. These events could be blocked by bcl-2, which upregulated themitochondrial K/H-exchanger, leading to increased removal of K. Inaddition, Bcl-2 and tBid proteins may counter-regulate mitochondrialpotassium transport. By removing/eliminating excess free radicalsantioxidants may restore mitochondrial function. Antioxidants actthrough a variety of mechanisms. The at least one scavenger of singletoxygen and the donor antioxidant are two different classes ofantioxidants that act through different mechanisms. The thirdantioxidant, while typically a scavenger of singlet oxygen, may be adifferent antioxidant that acts through a different mechanism.Scavengers of singlet oxygen reduce free radicals that contribute toinner ear pathology and thus to tinnitus. These free radicals may alsocause side effects of antibiotic treatment such as kidney damage andloss of balance. More specifically, by reducing free radicals, thescavengers of singlet oxygen prevent, among other damaging effects, thesinglet oxygen from reacting with lipids to form lipid hydroperoxides.Lipid hydroperoxides may play a role in causing tinnitus.

Even within the class of scavengers of singlet oxygen, it is believedthat various antioxidants react at different sites within the body, andin particular, within cells to attenuate free radical formation. Forexample, one of the scavengers of singlet oxygen is typically vitamin A.In various non-limiting embodiments described herein, the terminologyVitamin A and beta-carotene may be used interchangeably. However, theseembodiments in no way limit this disclosure. Vitamin A is a generic termthat captures a number of molecules with a biological activity ofretinol or carotenoids. Primary dietary forms of vitamin A/retinolinclude retinol esters and beta-carotene. The beta-carotene is made upof a polyene chain of 11 conjugated double bonds with methyl branchesspaced along the polyene chain, capped at both ends by cyclohexenylrings with 1,1,5-trimethyl substitution. Other forms of vitamin Ainclude xanxthophylls, astaxanthin, canthxanxin, lutein, and zeaxanthin,which include a backbone of beta-carotene with hydroxyl and/or carbonylsubstitution on one or more of the cyclohexenyl rings. For purposes ofthe subject disclosure, the vitamin A is typically present asbeta-carotene. Beta-carotene is a powerful scavenger of singlet oxygen,as well as nitric oxide and peroxynitrite, and may also scavenge lipidperoxyl radicals within a lipophilic compartment of a mitochondrialmembrane. Beta-carotene is an excellent scavenger of free radicals undernormal physiological conditions present in most tissues.

In addition to vitamin A, other scavengers of singlet oxygen may also bepresent in the composition of the subject disclosure. For example,another scavenger of singlet oxygen that may be present is resveratrol.Resveratrol is more efficient at scavenging hydroxyl radicals thanvitamin C, and the addition of resveratrol to the vitamins A may haveadditive effects.

The at least one scavenger of singlet oxygen may be present in thecomposition in a biologically effective amount. For purposes of thesubject disclosure, the biologically effective amount may be furtherdefined as an amount that is sufficient to produce an additive effect ina reduction in stress induced threshold shift or tinnitus when used incombination with other antioxidants and the magnesium. Additive effect,as used herein, refers to an effect that is equal to or greater than asum of the effects of the individual components. In order to produceadditive effect and the reduction in threshold shift or tinnitus, the atleast one scavenger of singlet oxygen is typically present in thecomposition in a total amount of at least 830 international units (IU),more typically from 830 to 120,000 IU, most typically from about 2,100to 70,000 IU for an adult dosage.

The amount of the vitamin A present in the composition is dependent uponthe form of vitamin A that is used. For example, in one embodiment,vitamin A is present as retinol in an amount of at least 830 IU, moretypically from 830 to 10,000 IU, more typically from 2,100 to 10,000 IU,most typically from 2,100 to 8,000 IU. As known in the art, a conversionof IU to weight for vitamin A (as retinol) is 3.33 IU/μg. Thus, at least830 international units (IU) of vitamin A (as retinol) is equivalent toat least 0.25 mg of vitamin A, from 830 to 10,000 IU of vitamin A (asretinol) is equivalent to from 0.25 to 3 mg of vitamin A, and from 2,100to 8,000 IU of vitamin A (as retinol) is equivalent to from 0.63 to 2.4mg vitamin A.

Alternatively, the vitamin A may be present in the composition asbeta-carotene, as opposed to retinol. The retinol activity equivalents(RAE) for retinol conversion to beta-carotene, which is a pro-vitamin Acarotenoid, is 1 mg to 12 mg. In terms of conversion of the amounts setforth above for the vitamin A present in the composition as retinol tothe vitamin A present in the composition as beta-carotene, in oneexample, a total amount of at least 3.0 mg or at least 830 internationalunits (IU) of vitamin A as beta-carotene, more typically from 3.0 to 180mg or 830 to 50,000 IU vitamin A as beta-carotene, most typically fromabout 7.2 to 108 mg or 2000 to 30,000 IU of vitamin A as beta-caroteneis typically present for an adult dosage. In another example, a totalamount of at least 3.0 mg or at least 10,000 international units (IU) ofvitamin A as beta-carotene, more typically from 3.0 to 36 mg or 10,000to 120,000 IU vitamin A as beta-carotene, most typically from about 7.5to 21 mg or 25,000 to 70,000 IU of vitamin A as beta-carotene istypically present for an adult dosage.

Specific amounts of the vitamin A present in the composition may bedependent on the body weight of the mammal. In one specific example, theamount of vitamin A present as retinol in the composition is about0.0178 mg/kg body weight. Thus, for an average human weighing about 70kg, the amount of vitamin A present as retinol in the composition may beabout 1.25 mg. If the vitamin A is in the form of beta-carotene, in oneexample, the beta carotene in the composition is about 0.257 mg/kg bodyweight may be present in an amount of about 18 mg. In another example,the beta-carotene in the composition may be about in an amount of about15 mg.

It is to be appreciated that, when additional scavengers of singletoxygen such as resveratrol are present in the composition in addition tovitamin A, the total amount of scavengers of singlet oxygen may begreater than the ranges set forth above for the at least one scavengerof singlet oxygen, so long as at least one scavenger of singlet oxygenis present in the amounts set forth above. In addition, other scavengersof singlet oxygen may be used in place of vitamin A, so long as theamount of the at least one scavenger of singlet oxygen is present withinthe amounts set forth above. When present, the resveratrol is typicallyincluded in the composition in an amount of at least 1 mg, moretypically in an amount of from 10 mg to 1500 mg, most typically in anamount of from 15 mg to 1000 mg.

Whereas the at least one scavenger of singlet oxygen tends to preventthe initial formation of lipid peroxides, the donor antioxidant tends toreduce peroxyl radicals and inhibits propagation of lipid peroxidationthat contributes to inner ear pathology and tinnitus. More specifically,the donor antioxidant reacts with and reduces peroxyl radicals and thusserves a chain-breaking function to inhibit propagation of lipidperoxidation. As is evident from the chain-breaking function of thedonor antioxidant in lipid peroxidation, the donor antioxidant functionswithin cell membranes. A specific donor antioxidant that is contemplatedfor use in the composition of the subject disclosure is vitamin E.Vitamin E is a generic term for all tocols and tocotrienol derivativeswith a biological activity of alpha-tocopherol. Primary dietary forms ofvitamin E include vitamin E itself and alpha-tocopherol. Trolox®, awater-soluble analogue of alpha-tocopheral commercially available fromHoffman-Laroche, Ltd. of Basel, Switzerland, is a research agent that istypically used as a source of vitamin E.

The donor antioxidant is typically present in the composition, forexample, in an amount of at least 75 IU, more typically from 75 IU to2,000 IU, more typically from 150 to 1,500 IU, most typically from 150IU to 800 IU. In another example, the donor antioxidant is present inthe composition in an amount of at least 75 IU, more typically from 75IU to 1,500 IU, most typically from 150 IU to 800 IU. As known in theart, a conversion of IU to weight for synthetic vitamin E is 0.66 mg/IUand for natural vitamin E is 0.45 mg/IU. Thus, when the donorantioxidant is synthetic vitamin E, in on example, at least 75 IU ofvitamin E is equivalent to at least 50 mg of vitamin E, from 75 to 2,000IU of synthetic vitamin E is equivalent to from 50 to 1,320 mg ofvitamin E, from 150 to 1,500 IU of synthetic vitamin E is equivalent tofrom 100 to 1,000 mg of vitamin E, and from 150 to 800 IU of syntheticvitamin E is equivalent to from 100 to 536 mg of vitamin E. In anotherexample, when the donor antioxidant is vitamin E, at least 75 IU ofvitamin E is equivalent to at least 50 mg of vitamin E, from 75 to 1500IU of vitamin E is equivalent to from 50 to 1000 mg of vitamin E, andfrom 150 to 800 IU of vitamin E is equivalent to from 150 to 600 mg ofvitamin E. As with the amount and type of vitamin A, specific amounts ofthe vitamin E present in the composition may be dependent on the bodyweight of the mammal. In one specific example, the amount of syntheticvitamin E present in the composition is about 3.8 mg/kg body weight.Thus, for an average human weighing about 70 kg, the amount of vitamin Epresent in the composition may be about 266 mg. In another specificexample, the amount of synthetic or natural vitamin E present in thecomposition is about 2.6 mg/kg body weight. Thus, for an average humanweighing about 70 kg, the amount of vitamin E present in the compositionmay be about 182 mg.

In addition to the at least one scavenger of singlet oxygen and thedonor antioxidant, the composition further includes the thirdantioxidant. While the third antioxidant may be a scavenger of singletoxygen, the third antioxidant may also be an antioxidant that functionsthrough a different mechanism. When the third antioxidant is a scavengerof singlet oxygen, the at least one scavenger of singlet oxygen is stillpresent in the composition as a separate component from the thirdantioxidant, and is still present in the composition in the amounts setforth above for the at least one scavenger of singlet oxygen. As aresult of the third antioxidant being another scavenger of singletoxygen, the resulting composition would have at least two scavengers ofsinglet oxygen.

The third antioxidant is typically vitamin C, which is a scavenger ofsinglet oxygen and reactive nitrogen species. It is to be appreciatedthat, although the third antioxidant is typically vitamin C, otherantioxidants may be used in place of the vitamin C, and the otherantioxidants may function through different mechanisms than vitamin C.The term vitamin C applies to substances that possess antiscorbuticactivity and includes two compounds and their salts: L-ascorbic acid(commonly called ascorbic acid) and L-dehydroascorbic acid. In additionto being known as ascorbic acid and L-ascorbic acid, vitamin C is alsoknown as 2,3-didehydro-L-threo-hexano-1,4-lactone,3-oxo-L-gulofuranolactone, L-threo-hex-2-enonic acid gamma-lactone,L-3-keto-threo-hexuronic acid lactone, L-xylo-ascorbic acid andantiscorbutic vitamin. Vitamin C is known to scavenge both reactiveoxygen species and reactive nitrogen species. It can be oxidized by mostreactive oxygen and nitrogen species, including superoxide, hydroxyl,peroxyl and nitroxide radicals, as well as such non-radical reactivespecies as singlet oxygen, peroxynitrite and hypochlorite. Vitamin Cthus inhibits lipid peroxidation, oxidative DNA damage, and oxidativeprotein damage.

In contrast to vitamin A, which functions best under conditions presentin most tissues, water-soluble vitamin C is an excellent free radicalscavenger in an aqueous phase to thus reduce free radicals at a sitedifferent from that of vitamin A. More specifically, ascorbic acidfunctions to reduce free radicals in fluid, such as in cytoplasmic fluidand/or blood, before the free radicals reach cell membranes.

The third antioxidant is typically present, for example, in an amount ofat least 4,000 IU, more typically from 4,000 to 60,000, more typicallyfrom 8,000 to 40,000 IU, most typically from 8,000 to 20,000 IU. Inanother example, the third antioxidant is typically present in an amountof at least 4,000 IU, more typically from 6,000 to 40,000 IU, and mosttypically from 8,000 to 20,000 IU. Using vitamin C as an example forconverting IU to weight units for the third antioxidant, as known in theart, a conversion of IU to weight for vitamin C is 0.05 mg/IU. Thus, atleast 4,000 IU of vitamin C is equivalent to at least 200 mg of vitaminC, from 6,000 to 60,000 IU of vitamin C is equivalent to from 300 to3,000 mg vitamin C, from 6,000 to 40,000 IU of vitamin C is equivalentto from 300 to 2,000 mg, from 8,000 to 40,000 IU of vitamin C isequivalent to from 400 to 2,000 mg vitamin C, and from 8,000 to 20,000IU vitamin C is equivalent to from 400 to 1,000 mg vitamin C. As withvitamins A and E, specific amounts of the vitamin C or other thirdantioxidant present in the composition may be dependent on the bodyweight of the mammal. In one specific example, the amount of vitamin Cpresent in the composition is about 7.14 mg/kg body weight. Thus, for anaverage human weighing about 70 kg, the amount of vitamin C present inthe composition may be about 500 mg.

As set forth above, the composition further includes a vasodilator.Typically, the vasodilator includes magnesium; however, the vasodilator,for purposes of the subject disclosure, may include other vasodilatorsin place of or in addition to magnesium, in place of or in addition tothose including magnesium, or may include only magnesium or onlymagnesium-containing compounds. Vasodilators can be used for treatingtinnitus. Vasodilators including magnesium prevent decreases in cochlearblood flow and oxygenation via biochemical mechanisms involving changesin calcium concentration and prostaglandins. Deficient cochlear bloodflow and lack of oxygenation can contribute to tinnitus by causingmetabolic changes in lateral wall tissues important for maintainingnormal homeostasis of the inner ear, e.g. endocochlear potential, andnormal transduction; and may cause cell death in sensitive hair cellswithin a cochlea of the ear. Vasodilators including magnesium have alsobeen found to improve the efficacy of immunosuppressant therapy orcarbogen inhalation therapy in recovery from sudden tinnitus.Furthermore, it has been found that magnesium deficiency leads toincreased calcium channel permeability and greater influx of calciuminto cochlear hair cells and afferent nerve endings, increased glutamaterelease, and auditory nerve excitotoxicity, each of which play a role inhealth of the inner ear. Although the vasodilators are known in the artfor treating tinnitus, the vasodilators, especially those includingmagnesium, exhibit an unexpected additive effect when combined with thebiologically effective amounts of the at least one scavenger of singletoxygen, the donor antioxidant, and the third antioxidant, especiallywhen the at least one scavenger of singlet oxygen is vitamin A, thedonor antioxidant is vitamin E, and the third antioxidant is vitamin Cfor purposes of treating noise-induced tinnitus. The additive effectreferred to above is greater than not only the most efficacious of thecomponents for treating inner ear pathology that causes tinnitus, buttypically greater than the sum of the effects of each of the componentsfor treating tinnitus. While vasodilators other than those includingmagnesium are envisioned for purposes of the present disclosure,additive effects are not observed with all vasodilators. For example,betahistine, which is another known vasodilator, does not exhibit anadditive effect.

The vasodilator including magnesium typically includes a magnesium saltor magnesium salt complex and, more specifically, magnesium sulfate ormagnesium citrate. Other vasodilators including magnesium that may besuitable for purposes of the subject disclosure include; magnesiumacetate, magnesium aspartate, magnesium carbonate, magnesium chloride,magnesium fumarate, magnesium gluconate, magnesium glycinate, magnesiumhydroxide, magnesium lactate, magnesium oxide, magnesium salicylate,magnesium stearate, and magnesium sulfate. Other representative saltsinclude but are not limited to; hydrobromide, hydrochloride, bisulfate,nitrate, arginate, ascorbate, oxalate, valerate, oleate, palmitate,laurate, borate, benzoate, phosphate, tosylate, maleate, fumarate,succinate, taurate, tartrate, naphthylate, mesylate, glucoheptonate,lactobionate and laurylsulphonate salts.

Typically, the vasodilator is present in the composition in an amount ofat least 50 mg. For example, when the vasodilator is magnesium, themagnesium is typically present in an amount of from 50 to 450 mg, mosttypically from 100 to 350 mg. As with vitamins A, C, and E, specificamounts of the vasodilator present in the composition may be dependenton the body weight of the mammal. In one specific example, the amount ofthe vasodilator including magnesium present in the composition is about4.46 mg/kg body weight. Thus, for an average human weighing about 70 kg,the amount of the vasodilator including magnesium present in thecomposition may be about 312 mg. In another example, the amount of thevasodilator including magnesium present in the composition is about 2.14mg/kg body weight. Thus, for an average human weighing about 70 kg, theamount of the vasodilator including magnesium present in the compositionmay be about 150 mg.

Non-limiting examples of amounts of the typical components included inthe composition, along with more and most typical amounts, aresummarized in Table 1 below.

TABLE 1 More Most Typical Typical Typical Dosage, mg/kg Component AmountAmount Amount body weight Vitamin A ≧830 IU 830-10,000 IU 2100-8,000 IU0.0178 mg/kg Vitamin ≧830 IU 830-50,000 IU 2,000-30,000 IU 0.257 mg/kg AAs beta- carotene Vitamin C ≧4,000 IU 4,000-60,000 IU 8,000-20,000 IU7.14 mg/kg Vitamin E ≧75 IU 75-2000 IU 150-800 IU 3.8 mg/kg (synthetic)Magnesium ≧50 mg 50-450 mg 100-350 mg 4.46 mg/kg

With respect to Table 1, the amounts specified for the antioxidants andthe vasodilator correlate, in terms of biological effectiveness, toamounts used for humans. Furthermore, it is to be appreciated that thebiologically effective amounts of the antioxidants and vasodilator maybe lower within the above ranges for children than for the averagehuman, based on lower US recommended daily allowances and maximum intakelevels for children. This is evident based on the typical dosages inTable 1 based on mg/kg.

Other non-limiting examples of amounts of the typical componentsincluded in the composition, along with more and most typical amounts,are summarized in Table 2 below.

TABLE 2 More Most Typical Typical Typical Dosage, mg/kg Component AmountAmount Amount body weight Vitamin A ≧830 IU 830-120,000 IU 2,100-70,000IU — Vitamin ≧830 IU 830-50,000 IU 2,100-5,900 IU 0.0178 mg/kg A AsRetinol Vitamin ≧10,000 IU 10,000-120,000 IU 25,000-70,000 IU 0.214mg/kg A As beta- carotene Vitamin C ≧4,000 IU 6,000-40,000 IU8,000-20,000 IU 7.14 mg/kg Vitamin E ≧75 IU 75-1,500 IU 150-800 IU 2.6mg/kg Magnesium ≧50 mg 50-450 mg 100-350 mg 2.14 mg/kg

With respect to Table 2, the amounts specified for the antioxidants andthe vasodilator correlate, in terms of biological effectiveness, toamounts used in animal studies on guinea pigs. Furthermore, it is to beappreciated that the biologically effective amounts of the antioxidantsand vasodilator may be lower within the above ranges for children thanfor the average human, based on lower U.S. recommended daily allowancesand maximum intake levels for children. This is evident based on thetypical dosages in Table 2 based on mg/kg.

In addition to the antioxidants and vasodilator, other components mayalso be present in the composition for treating tinnitus. Thesecomponents may be used for treating the side effects of the antibiotictreatment also. For example, in one embodiment, the composition furtherincludes a withanolide. Withanolides have been suggested for use inanti-inflammatory, anti-tumor, cytotoxic, and immunologicalapplications. One example of a specific withanolide that may be includedin the composition of the subject disclosure is the withanolideextracted from day lily plants. The extract is a powerful naturalantioxidant which may be effective in preventing cell death in the innerear by interrupting the cell-death pathway initiated by deafferentationof the auditory nerve. When included in the composition, the withanolidemay be present in an amount of at least 10 ppm, more typically from 10to 1000 ppm. Additional components, besides withanolides, can also beincluded. Typically, the composition is free of components thatinterfere with the biological mechanisms through which the at least onescavenger of singlet oxygen, the donor antioxidant, the thirdantioxidant, and the vasodilator function. The composition is alsotypically free of additional components that could degrade or neutralizethe at least one scavenger of singlet oxygen, the donor antioxidant, thethird antioxidant, and the vasodilator function when mixed therewithprior to internally administering the composition to the mammal. Thoseof skill in the art can readily identify such components in view of themechanisms by which the individual components in the compositionfunction as set forth above (e.g., components that causevasoconstriction, various oxidizing agents, etc.).

It is also to be appreciated that, even if additional components arepresent in the composition that could interfere with the mechanisms bywhich the at least one scavenger of singlet oxygen, the donorantioxidant, the third antioxidant, and the vasodilator function, thecomposition described above is may still be effective for purposes oftreating side effects of the antibiotic treatment. As one example, andas described in further detail below, the composition including the atleast one scavenger of singlet oxygen, the donor antioxidant, the thirdantioxidant, and the vasodilator may be effective for treating tinnitusand other side effects of antibiotic treatment when administered inconjunction with aminoglycoside antibiotics. This is true even thoughaminoglycoside antibiotics, themselves, are responsible for causingtinnitus and other side effects such as kidney damage and loss ofbalance through free radical formation.

In view of the fact that components as detrimental as aminoglycosidescan be administered in conjunction with the composition described above,it is clear that additional components that are less detrimental to thespecific mechanisms by which the at least one scavenger of singletoxygen, the donor antioxidant, the third antioxidant, and thevasodilator function can also be present in the composition with anexpectation that the composition maintains effectiveness for purposes oftreating side effects of antibiotic treatment such as antibiotic-inducedtinnitus. Examples of some additional components that may be included inthe composition include, but are not limited to, excipients, flavoringagents, fillers, binders, and additional vitamins or minerals notspecifically mentioned herein.

As alluded to above, the method of treating tinnitus and the method oftreating side effects from antibiotic treatment of the instantdisclosure includes the step of internally administering the compositionof the subject disclosure to a mammal. The composition may be orallyadministered to the mammal, such as in the form of a tablet, liquid,gel, etc. Alternatively, the composition may be intravenouslyadministered to the mammal through an IV or an injection of thecomposition. When specifically used to treat antibiotic-inducedtinnitus, the composition may also be locally administered via the roundwindow membrane of the cochlea. As a specific example, the vitamins A,C, and E, the vasodilator including magnesium, and the optionalcomponents may be first combined to form the composition, with thecomposition then administered to the mammal. Alternatively, the vitaminsA, C, and E, the vasodilator including magnesium, and the other optionalcomponents may be separately administered, in which case the compositionforms within the mammal.

For purposes of the subject disclosure, tinnitus is typically associatedwith stress (noise, antibiotic, genetic)-induced hearing loss asobjectively measured in terms of differences in threshold shift, orthrough measurement of a percentage of hair cell loss. In guinea pigstudies, tinnitus, as objectively measured as a change in Gap-Detection,is observed in the majority of animals, either transiently or prolonged,following exposure to noise. Reduction in tinnitus reflecting theefficacy of the composition for treating noise-induced tinnitus may bemeasured as an average difference in threshold shift from baselinethreshold sensitivity at 4, 8, and 16 kHz, as compared to an untreatedcontrol, after exposure to high indensity Octave Band Noise centered at4 kHz for three hours. Small differences in threshold shift may beassociated with less tinnitus and greater efficacy of the compositionfor treating the antibiotic-induced tinnitus.

For purposes of the subject disclosure, tinnitus may be objectivelymeasured in terms of differences in threshold shift, or throughmeasurement of a percentage of hair cell loss. In guinea pig studies,tinnitus and the efficacy of the composition for treatingantibiotic-induced tinnitus may be measured as an average difference inthreshold shift from baseline threshold sensitivity at 4, 8, and 16 kHz,as compared to an untreated control, after exposure to 120 decibel SPLOctave Band Noise centered at 4 kHz for five hours. Larger differencesin threshold shift correlate to less tinnitus and greater efficacy ofthe composition for treating the antibiotic-induced tinnitus.

It is has been shown that hair cell loss correlates to threshold shift.For example, in guinea pig ears that recover from temporary thresholdshift, morphological damage is limited to tips of stereocilia in a thirdrow of outer hair cells (OHCs) whereas ears from animals with permanentthreshold shift have damage to all three rows of OHCs and, in somecases, the inner hair cells (IHCs), with damage throughout the length ofthe stereocilia as well as the to the body of the hair cell.

In one embodiment, the composition of the present disclosure isadministered to the mammal within three days of trauma to the inner earof the mammal in order to alleviate permanent threshold shift. It is tobe appreciated that by administering the composition within three daysof trauma, treatment prior to trauma is also contemplated through themethod of the present disclosure. Data from animal studies indicate thattemporary threshold shift measured 24 hours post-trauma iswell-correlated with permanent threshold shift. Given the relationshipbetween temporary threshold shift and permanent threshold shift, it isclinically beneficial to reduce temporary threshold shift. As such, thecomposition is typically administered within one day of trauma to theinner ear of the mammal. Even so, it is expected that treatment withinthree days with the composition of the present disclosure issubstantially as effective in minimizing permanent threshold shift astreatment within one day.

Treatment within three days is most appropriate when the mammal hassustained trauma to the inner ear through unexpected loud noise or othertrauma. Ideally, the composition is administered to the mammal prior totrauma to the inner ear. Treatment prior to trauma is most feasible whenthe mammal is preparing for sustaining trauma to the inner ear. Forexample, the composition may be administered prior to restorationsurgery performed on the inner ear. As another example, if a person willbe firing a weapon or attending an event such as a rock concert, theperson may begin treatment prior to sustaining the trauma to the innerear to attain the best results.

After initial administration of the composition, the composition istypically administered to the mammal each day for at least five daysfollowing the trauma to the inner ear of the mammal. Although excellentresults have been achieved through such treatment, it is to beappreciated that other treatment regimens may also prove efficacious forpurposes of the present disclosure.

For the method of treating side effects of antibiotic treatment, thecomposition is internally administered to the mammal in conjunction withadministration of the antibiotic. In this regard, the method alsoincludes the step of internally administering the antibiotic, whichantibiotic is capable of inducing tinnitus in the mammal. It is to beappreciated that, even though the antibiotic with which the compositionis administered is capable of inducing tinnitus, the method of theinstant disclosure is not strictly limited to treatment of tinnitus thatis induced by the antibiotics. More specifically, the method of theinstant disclosure proscribes the step of administering the subjectcomposition in conjunction with administration of the antibiotic for anypurpose including for treating any side effect of the antibioticsincluding not only antibiotic-induced tinnitus, but also kidney damage,loss of balance, among other side effects.

Non-limiting examples of antibiotics that are thought to potentiallycontribute to inducing tinnitus in mammals include aminoglycosideantibiotics such as amikacin, arbekacin, gentamicin, kanamycin,neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin,tobramycin, and apramycin; and glycopeptide antibiotics such asvancomycin, teicoplanin, telavancin, bleomycin, ramoplanin, anddecaplanin. The treatment is particularly effective againstaminoglycoside-induced side effects. In one specific embodiment, theantibiotic is gentamicin, which is commonly used in developing countriesdue to low cost and effectiveness against certain drug-resistantdiseases.

The dosages of the antibiotic that are administered to the mammal mayvary within the medically-accepted ranges for therapeutic treatment, asmay the number of days over which the antibiotic is administered. Theantibiotic is typically administered daily. For purposes of objectivelyestablishing the effectiveness of the treatment described herein, adosage of 140 g of gentamicin was administered to guinea pigs for aperiod of 16 days. However, it is to be appreciated that the specificdosages and days over which the antibiotic is administered are notmaterial for purposes of establishing the viability of the treatmentdescribed herein so long as the composition described herein isadministered in conjunction with administration of the antibiotic(although the effectiveness of the treatment described herein may varybased upon dosages and days over which the antibiotics areadministered).

For purposes of the instant disclosure, administration of thecomposition described herein “in conjunction with” administration of theantibiotic refers to a connection in the administration of thecomposition and the antibiotic during the course of antibiotictreatment. To maximize effectiveness of the treatment described herein,it is desirable to establish stable serum levels of the at least onescavenger of singlet oxygen, the donor antioxidant, the thirdantioxidant, and the vasodilator at the time that the antibiotics reachserum levels within the mammal that may cause material side effects suchas tinnitus. Typically, the composition is internally administered tothe mammal no longer than three days after administration of a firstdosage of the antibiotic, which is sufficient to achieve the stableserum levels of the at least one scavenger of singlet oxygen, the donorantioxidant, the third antioxidant, and the vasodilator before theantibiotics begin to materially cause the side effects such as tinnitus.In one embodiment, the composition is internally administered prior toadministration of the first dosage of the antibiotic. In thisembodiment, the composition may be administered at least five days,alternatively at least ten days, prior to administration of the firstdosage of the antibiotic for purposes of maximizing the effectiveness ofthe treatment for side effects of antibiotic treatment described herein.

Once administration of the composition has begun, the composition istypically administered each day the antibiotic is administered tomaintain adequate serum levels of the at least one scavenger of singletoxygen, the donor antioxidant, the third antioxidant, and thevasodilator. Additionally, the composition is typically administered forat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or 21 days following cessation of administration of theantibiotic. This is typically performed for purposes of ensuring thatadequate serum levels of the at least one scavenger of singlet oxygen,the donor antioxidant, the third antioxidant, and the vasodilator aremaintained until the serum levels of the antibiotic decrease, andtypically until all of the additional free radicals formed secondary tothe antibiotic treatment are eliminated.

In varying embodiments, an average difference in threshold shift inmammals from baseline threshold sensitivity at 4, 8, and 16 kHz, ascompared to an untreated control, is at least 25 decibels after exposureto 120 decibel SPL Octave Band Noise centered at 4 kHz for five hours.More specifically, the threshold shift in mammals treated with thecomposition of the present disclosure is expected to be at least 25decibels lower than the threshold shift in mammals that are treated witha control saline solution. To obtain those results, the composition isorally administered one hour prior to exposure to the noise andadministered again each day for five days subsequent to the exposure tothe noise. The threshold shift is measured 10 days after exposure to thenoise using auditory brainstem response (ABR) testing. Similar resultswould be anticipated using other alternative measures of auditory orsensory cell function, such as psychophysical tests or otoacousticemission measures. While effectiveness of treatment in accordance withthe method of the instant disclosure is related to measurement andmitigation of shifts in threshold, which is routinely associated withtinnitus, it is to be appreciated that the method is not strictlylimited to treatment of noise-tinnitus alone.

In addition, outer hair cell loss and inner hair cell loss is measuredboth in the whole cochlea and in a trauma region of the cochlea. Aftertreatment according to the method of the present disclosure, outer haircell loss in the whole cochlea is typically less than 10%, and innerhair cell loss in the whole cochlea is typically less than 5%. Outercell hair loss in the trauma region is typically less than 20%, whileinner hair cell loss in the trauma region is typically less than 10%.For the method of treatment in accordance with the present disclosure,outer hair cell loss in the whole cochlea is typically less than 50%,while inner hair cell loss in the whole cochlea is typically less than5%. To obtain those results, the composition is orally administered toguinea pigs for 10 to 16 days prior to first administration of theantibiotic, and the composition and antibiotic are administered againeach day for forty days. The guinea pigs are then euthanized and thehair cells counted. Conversely, when the antibiotic is administered forforty days without administering the composition described herein inconjunction with the antibiotic, outer hair cell loss approaches 80%,while inner hair cell loss is about 8%.

One or more of the values described above may vary by −5%, −10%, −15%,−20%, −25%, etc. so long as the variance remains within the scope of thedisclosure. Unexpected results may be obtained from each member of aMarkush group independent from all other members. Each member may berelied upon individually and or in combination and provides adequatesupport for specific embodiments within the scope of the appendedclaims. The subject matter of all combinations of independent anddependent claims is herein expressly contemplated. The disclosure isillustrative including words of description rather than of limitation.Many modifications and variations of the present disclosure are possiblein light of the above teachings, and the disclosure may be practicedotherwise than as specifically described herein. In additionalnon-limiting embodiments, all values and ranges of values within anyaforementioned range of numbers are hereby expressly contemplated.

What is claimed is:
 1. A method of treating tinnitus, said methodcomprising the step of internally administering a composition to themammal, wherein the composition consists essentially of a biologicallyeffective amount of vitamin A, vitamin E, vitamin C, a vasodilatorcomprising magnesium, and, optionally, a withanolide, and/orresveratrol.
 2. A method as set forth in claim 1 wherein the step ofinternally administering the composition is further defined as orallyadministering the composition to the mammal.
 3. A method as set forth inclaim 1 wherein the step of internally administering the composition isfurther defined as intravenously administering the composition to themammal.
 4. A method as set forth in claim 1 further comprising the stepof detecting tinnitus in a mammal.
 5. A method as set forth in claim 4wherein the step of internally administering the composition occursprior to the step of detecting the tinnitus in the mammal.
 6. A methodas set forth in claim 5 wherein an average difference in threshold shiftin mammals from baseline threshold sensitivity at 4, 8, and 16 kHz, ascompared to an untreated control, is at least 25 decibels when traumaresults from exposure to 120 decibel SPL Octave Band Noise centered at 4kHz for five hours.
 7. A method as set forth in claim 1 wherein thecomposition further comprises resveratrol.
 8. A method as set forth inclaim 1 wherein the vitamin A is present in the composition in an amountof at least 830 IU.
 9. A method as set forth in claim 1 wherein thevitamin C is present in the composition in an amount of at least 4,000IU.
 10. A method as set forth in claim 1 wherein the vitamin E isfurther defined as a water-soluble analogue of alpha-tocopheral.
 11. Amethod as set forth in claim 1 wherein the vitamin E is present in thecomposition in an amount of at least 75 IU.
 12. A method as set forth inclaim 1 wherein the composition further comprises the withanolide.
 13. Amethod as set forth in claim 1 wherein the vasodilator is present in anamount of at least 50 mg.
 14. A method as set forth in claim 1 whereinthe composition provides an additive effect that is equal to or greaterthan a sum of the effects of the individual components.
 15. A method oftreating tinnitus, said method comprising the step of internallyadministering a composition to the mammal, wherein the compositionconsists essentially of a biologically effective amount of vitamin A,vitamin E, vitamin C, a vasodilator comprising magnesium, and,optionally, a withanolide, and/or resveratrol, and wherein thecomposition is administered after peroxyl radical formation.
 16. Amethod as set forth in claim 15 wherein the formation of peroxylradicals is further defined as oxidative DNA damage.
 17. A method as setforth in claim 15 wherein the formation of peroxyl radicals is furtherdefined as oxidative protein damage.
 18. A method as set forth in claim15 wherein the composition provides an additive effect that is equal toor greater than a sum of the effects of the individual components.
 19. Amethod of treating tinnitus, said method comprising the step ofinternally administering a composition to the mammal, wherein thecomposition consists essentially of a biologically effective amount ofvitamin A, vitamin E, vitamin C, a vasodilator comprising magnesium,and, optionally, a withanolide, and/or resveratrol, and wherein thecomposition is administered after lipid peroxidation in the mammal. 20.A method as set forth in claim 19 wherein the composition provides anadditive effect that is equal to or greater than a sum of the effects ofthe individual components.
 21. A method of treating tinnitus, saidmethod comprising the step of internally administering a composition tothe mammal, wherein the composition consists essentially of abiologically effective amount of vitamin A, vitamin E, vitamin C, avasodilator comprising magnesium, and, optionally, a withanolide, and/orresveratrol, and wherein the composition is administered aftervasoconstriction of blood vessels in an ear of the mammal.
 22. A methodas set forth in claim 21 wherein the composition provides an additiveeffect that is equal to or greater than a sum of the effects of theindividual components.
 23. A method of treating tinnitus, said methodcomprising the step of internally administering a composition to themammal, wherein the composition consists essentially of a biologicallyeffective amount of vitamin A, vitamin E, vitamin C, a vasodilatorcomprising magnesium, and, optionally, a withanolide, and/orresveratrol, and wherein the composition is administered after formationof lipid peroxyl radicals within a lipophilic compartment of amitochondrial membrane in the mammal.
 24. A method as set forth in claim23 wherein the composition provides an additive effect that is equal toor greater than a sum of the effects of the individual components.